Project Summary/Abstract DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. The events that assemble the multi-enzyme replisomes that act at each replication fork are tightly regulated to ensure that each chromosome is replicated exactly once. Consistent with their importance, defects in or misregulation of replication initiation proteins are known to lead to cancer and developmental abnormalities. It is critical to understand how the essential event of replication initiation occurs and how this event leads to the appropriate assembly of the replication machinery. In recent years, significant advances have been made in our understanding of the initial event of this process, the selection of the sites of replication initiation (origins of replication) through the loading of the replicative DNA helicase. In contrast, we know significantly less about the events that activate these loaded helicases and the subsequent assembly of the DNA replication machinery. The proposed research will exploit a novel in vitro assay that recapitulates the events of DNA replication initiation from a defined origin of replication. We will use this assay and new assays derived from it to address fundamental questions concerning the initiation of replication. We will primarily focus on the committed step of replication initiation, DNA helicase activation. In specific aim one, we will develop assays to address how the loaded helicase is primed for helicase activation but maintained in an inactive state. In the second aim, we will use novel DNA substrates and assays to determine how the numerous proteins required for helicase activation contribute to this event, with a focus on the known helicase-activating protein, Cdc45. In the last aim, we will address how helicase activation and DNA unwinding are coordinated with the initiation of DNA synthesis to prevent the generation of excess single-stranded DNA and activation of DNA-damage checkpoint.